Project Summary/Abstract GATA6 and GATA4 are two members of the GATA family of zinc finger transcription factors and have been shown to be important for pancreatic development. Heterozygous mutations in GATA6 and GATA4 account for the majority of cases of patients with pancreatic agenesis. However, GATA6 and GATA4 clearly play a more complex role in pancreatic development and function as these same heterozygous mutations can result in diabetes of varying onset and severity in some individuals without pancreatic agenesis, differing even within family members carrying the same mutations. Factors that cause disease due to haploinsufficiency can be particularly important therapeutic targets as small modifications in their action could be the difference between disease and normal function. Therefore, understanding the role of the GATA factors will advance our knowledge of beta cell function. Though these proteins have been studied in mice, they do not recapitulate the human phenotype, making studies in human cells necessary. The development of techniques to differentiate human pluripotent stem cells (hPSCs) into pancreatic cells has allowed for the use of a human system in which to accomplish these investigations. Preliminary data from our group show that GATA6 is necessary for normal endoderm induction during differentiation of hPSCs, and that loss of GATA6 results in defective glucose-stimulated insulin secretion in differentiated pancreatic beta cells. Additionally, preliminary studies show that GATA6 and GATA4 expression can be induced by retinoic acid, one of the inductive signals involved in the differentiation of pancreatic cells, suggesting interaction between the two pathways. The research described in this proposal will use two human models of pancreatic beta cells, differentiated hPSCs and a human beta cell line, to characterize the role of GATA6 and GATA4 in glucose-stimulated insulin secretion and to define the interaction between the GATA factors and retinoic acid signaling. By selectively knocking down and overexpressing the GATA factors in mature pancreatic beta cells, we will be able to determine their effect on insulin secretion independent of their effect on development. Utilizing this same technique in both differentiating and mature pancreatic beta cells while modifying retinoic acid signaling, we will be able to characterize the interaction between GATA4/6 and retinoic acid. These studies will help advance our knowledge of pancreatic beta cell function and development and therefore allow for a better understanding of the pathophysiology underlying diseases resulting from their dysfunction, including diabetes.